For many clinical and research purposes, it is necessary to measure the concentration of ions, particularly cations such as lithium, ammonium, sodium, potassium, or calcium, in biological fluids such as serum, plasma, or urine.
In recent years, physiological electrolyte analyzers based on ion-selective electrode (ISE) technology have been used increasingly in the clinical laboratory and doctor's office for the determination of such ions as H.sup.+, Na.sup.+, K.sup.+, and Ca.sup.2+. These analyzers are as simple and as rapid to use as the familiar pH meter. In such analyzers, an ISE is used together with an external reference electrode in such a manner that they are simultaneously immersed in a sample solution. An electrical potential is developed between the electrodes which is related to the presence of the ion to which the ISE is sensitive. The potential is proportional to the logarithm of the ion concentration. The relationship between potential and the logarithm of the ion concentration is described by the Nernst equation. The intercept of the potential on the y-axis, i.e., the potential at an infinitesimal ion concentration, is referred to as the offset.
The traditional methods for monitoring lithium ion concentrations, however, have been atomic absorption spectroscopy and flame emission photometry. Although accurate and precise, these methods are time-consuming, require very expensive and cumbersome equipment, and are not particularly suited to automation. The time and expense required make such methods unsuitable for rapid monitoring of serum lithium concentration in a doctor's office, as should be done with patients receiving lithium.
Likewise, ammonia ion concentration has been measured by conductimetry and by the reaction with phenol in the presence of hypochlorite to form a chromogen, indophenol. These methods for ammonium measurement are not ideal as they are time-consuming and require complex equipment and several reagents.
Although the measurement of lithium and ammonia using ISE technology would be an improvement over these prior measurement techniques, the use of ISE technology has presented several challenges. For example, an ISE useful for lithium determination must meet several rigorous criteria. Most importantly, such an ISE must be highly selective for lithium over other cations found in serum, particularly sodium and potassium. The level of NA.sup.+ in serum is typically over 1400 times the lowest lithium levels of clinical importance. The ISE for lithium should also have good sensitivity so that concentrations of lithium in serum of about 0.2 mmol/L can be accurately determined, and, for the most accurate measurement, a plot of electrode potential against the logarithm of lithium concentration should be linear or nearly so in the concentration region of interest. The ISE must also be resistant to interference from organic molecules found in blood serum, especially lipoproteins and other proteins. Rapid and stable response to lithium ion is also highly desirable. Resistance to water hydration when exposed to aqueous samples is also extremely important when providing a lithium ISE useful commercially. The occurrence of hydration requires that electrodes be changed at intervals of only a few weeks or even days. This has proven to be a great obstacle to a commercially useable lithium ISE.
ISEs have been developed by T. Shono, using crown ethers as lithium-selective ionophores (K. Kimura et al., Anal. Chem. 59:2331-2334 (1987)), and by Dr. W. Simon in Zurich, using ionophores known as "neutral ligands" (EPO Publication No. 017452 A2; E. Metzger, Anal. Chem. 58:132-135 (1986); E. Metzger, Anal. Chem. 59:1600-1603 (1987)). While these publications demonstrate the principal that lithium-selective and neutral ligand ionophores are useful, the ISEs described are not suitable for clinical applications due to limited performance characteristics.
As part of the measurement circuit of an ISE, an internal reference electrode is required. In traditional prior ISEs, this was usually made with a silver/silver chloride electrode in contact with a liquid reference solution that was in turn in contact with the ion-selective membrane. In more recent designs, the liquid internal reference electrode has been replaced by a solid support in the form of an electrical element or substrate (U.S. Pat. No. 4,276,141 to Hawkins; U.S. Pat. No. 3,856,649 to Genshaw et al.). However, a primary cause for problems with known solid support ISEs is believed to be related to difficulties attributed to the physical adhesion between the ion selective layer and the electrical element or substrate. Such an element or substrate can be a wire or a semiconductor (ISEFET). Another form of such an element or substrate is a silver-silver chloride pellet made by pressurizing a silver and silver chloride powder mix at very high pressure. With each of these types of elements or substrates, however, there is believed to be insufficient porosity to retain the ion-selective layer or membrane in sufficiently good physical contact to insure good electrochemical interaction.
Even more recently, a polymeric membrane ISE design using graphite as the internal reference electrode has been described (U.S. Pat. No. 4,549,951 to Knudson et al.; U.S. Pat. No. 4,431,508 to Brown et al.). Of these two approaches, only Brown et al. describes pre-treating the graphite substrate to help stabilize drift, but even Brown et al. is unsatisfactory for routine clinical analysis because the offset, i.e., the intercept of the Nernst equation E.degree., varies from electrode to electrode in a broad range. This is undesirable because the offset, sometimes referred to as the asymmetric potential (AP), must match with the instrument on which the electrode is to be used, and mismatches can lead to analytical error.
Therefore, there is a need for an improved design of ISEs, particularly for the detection of lithium and ammonium, but also for detection of other cations such as sodium and potassium. Such ISEs should be selective and sensitive and respond rapidly. They should be resistant to interference from serum components such as proteins and lipids. They should provide for improved electrochemical interaction between the electrically conductive element and the sample through a polymeric ion selective layer. Most importantly, they should have a very narrow offset or AP range to improve matching of the electrode with the instrument with which it is to be used.